Novel functions of pulmonary endothelial and epithelial CXCR2 in leukocyte trafficking to infected lungs and in lung injury

• Applicant: Professor Dr. Alexander Zarbock
• Subject Area: Anaesthesiology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 491579569

Life threatening bacterial lung diseases are caused by excessive neutrophil recruitment and increased vascular permeability, both driven by the release of pro-inflammatory mediators. Increased permeability leads to alveolar flooding with protein-rich edema fluid. The resulting loss of gas exchange leads to acute respiratory failure and typically catastrophic illness, termed acute respiratory distress syndrome, requiring ventilatory and critical care support. Improved understanding of the underlying mechanism is of keen importance. Therefore, we aim to identify the exact role of CXCR2 on pulmonary epithelial and endothelial cells by analysing two putative functions of CXCR2: Influencing the vascular and alveolar permeability and regulation of chemokine transport. To address this, we will generate conditional mouse lines deficient for CXCR2 in epithelial or in endothelial cells, respectively, and investigate leukocyte recruitment following CXCL1 or CXCL2 introduction. Furthermore, we will analyse these mice for the transport of chemokines to the capillary lumen by intratracheal injection of biotin-labelled chemokines, which will be visualized by electron microscopy using streptavidin coated gold particles. Vascular permeability will be assessed by using in vitro transwell assays with endothelial cells, and later in vivo analysing Evan´s Blue leakage. To identify a possible function in vesicle-mediated transcytosis, we will investigate whether CXCR2 participates in the formation of clathrin-coated pits or caveolae by using biochemical methods. As second aim, we will identify the roles of endothelial and epithelial CXCR2 during bacterial lung infection. Here, we will analyse in detail leukocyte recruitment, vascular permeability, and chemokine expression in response to bacterial stimuli. In vivo lung microscopy will allow us to detect at which location recruited neutrophils are delayed during their course of emigration. As third aim, we will analyse the signalling pathways following CXCR2-activation, and identify the role of beta-arrestin as multifunctional adaptor molecule in this pathway. Using biochemical methods, we will identify the role of beta-arrestin in CXCR2-mediated localization, complex formation, as well as in cytokine expression. Furthermore, we hypothesise a role for beta-arrestin during trans-epithelial migration. Following in vitro-studies using shRNA against beta-arrestin, we will analyse in detail leukocyte recruitment, cytokine expression, and bacterial burden during lung infection in WT and beta-arrestin-deficient mice. Finally, to estimate whether the observed mechanisms are unique to pulmonary cells, we will investigate the role of endothelial CXCR2 during leukocyte adhesion and transmigration in the cremaster muscle by intravital microscopy. Taken together, these experiments will substantially broaden our knowledge concerning CXCR2-mediated leukocyte recruitment, cytokine transcytosis and vascular permeability.

• DFG Programme: Research Grants
• International Connection: Israel
• Cooperation Partner: Professor Dr. Ronen Alon, Ph.D.